Lung cancer is a growing global health concern (1), and cigarette smoking is a well-known risk factor for lung carcinogenesis (2). Nevertheless, approximately 25% of lung cancer cases are not attributable to tobacco smoking, and over 50% of female patients have been reported as never-smokers (3, 4). The prevalence of smoking among women in East Asia is lower compared to that in western countries (5). In Taiwan, more than 90% of female lung cancer patients are never-smokers (6, 7). Lung cancer in never smokers is considered a distinct disease entity with a proteogenomic landscape and oncogenic mechanisms different from those in smokers (8). Therefore, identifying genetic and environmental factors associated with lung cancer risk in never-smokers is urgently needed, especially in Asia.

Inflammation is considered one of the hallmarks of cancer, promoting tumorigenesis and neoplastic progression (9). Chronic infection and inflammation are strongly correlated with cancer risk (10). In addition to cigarette smoking, other environmental factors including chronic inflammation and particle/pollutant inhalation may also play a role in cancer developments (11, 12). However, limited data on the association between chronic infection/inflammation and lung carcinogenesis in never smokers are available.

Inflammation, including immune responses to chronic infection, may help to eliminate abnormal cells and prevent tumorigenesis (13, 14). However, tumors may overcome immune surveillance through mechanisms of immune evasion (9). The programmed death-ligand 1 (PD-L1) or programmed death-ligand 2 (PD-L2) on cancer cells would bind to programmed death-1 (PD-1) on immune cells, which could inhibit T cell activation and proliferation (15–19). PD-1 as a transmembrane protein, which is expressed on activated lymphocytes (T cells, B cells, and tumor specific T cells), natural killer cells, monocytes, and macrophages, involves in the tumorigenesis by restraining immune response (20, 21). The expression of PD-L1 is induced by oncogenes’ expression and various proinflammatory molecules and inhibited by the tumor suppressor genes expression, such as PTEN alternations (22). Therefore, activation of PD-1/PD-L1 pathway could lead to immune suppression and promote tumor growth in various cancer types (23, 24).

Further, high PD-L1 expression is not only found to accelerate skin carcinogenesis (25) but also associated with tumor differentiation, vascular invasion, and resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor treatment in non-small cell lung cancer (NSCLC) (26, 27). In NSCLC, the PD-1/PD-L1 pathway involving tumor proliferation and interacting with tumor microenvironment were greatly investigated. However, the role of PD-L2 in biological function of tumors was rarely studied.

Previous studies reported the association of PD-L1 gene polymorphisms with NSCLC risk mostly in smokers (28, 29). In never-smokers, the roles of PD-1, PD-L1, and PD-L2 gene polymorphisms in lung carcinogenesis remain unclear. Therefore, we conducted a case-control study in never-smoking women to explore the effects of PDCD1 (encoding PD-1), CD274 (encoding PD-L1), and PDCD1LG2 (encoding PD-L2) single nucleotide polymorphisms (SNPs) on lung carcinogenesis. The coding regions of these genes were of particular interest. In this study, chronic obstructive pulmonary disease (COPD), pulmonary tuberculosis (TB) infection, cooking fume exposure, and environmental tobacco smoking were defined as inflammation-related environmental exposures. We specifically investigated the interactions between these environmental factors and SNPs with regard to lung cancer susceptibility.

In this multi-center case-control study, a total of 12 PDCD1LG2 SNPs (rs2381282, rs4742103, rs4237162, rs4742104, rs12237624, rs78096119, rs6476988, rs7857315, rs10975178, rs7854413, rs56001683, and rs7858319) associated with LUAD risk in never-smoking women were identified. Among them, rs2381282, rs4742103, rs4237162, rs4742104, rs78096119, and rs56001683 were recognized as tagged SNPs. Furthermore, PDCD1LG2 SNPs rs12237624 and rs78096119 had significant associations with a history of pulmonary TB infection related to LUAD susceptibility. The PDCD1LG2 SNPs rs12237624, rs78096119, and rs2381282 were associated with PD-L2 expression via eQTL analysis. To our knowledge, this is the first study identifying novel PD-L2 gene polymorphisms associated with lung carcinogenesis in female never-smokers.

Among the 12 PDCD1LG2 SNPs, the clinical significance of rs7854413 was the most commonly reported in the previous literatures. A cohort study in south India reported that patients with the PDCD1LG2 SNP rs7854413 and lymphatic filariasis infection were susceptible to chronic lymphatic pathologies (40), and rs7854413 polymorphism was related to advanced fibrosis and development of hepatocellular carcinoma from patient with non-alcoholic steatohepatitis (41). Notably, rs7854413 was also associated with recurrence in patients with early-stage NSCLC (42). During the process of literature review, no studies on these PDCD1LG2 SNPs other than rs7854413 were reported. The functional role of these SNPs in lung carcinogenesis warrants further investigation.

Immune checkpoint blockade through inhibition of the PD-1/PD-L1 pathway is a state-of-the-art cancer immunotherapy (43). In contrast, the clinical significance of PD-L2 is seldom investigated (44). The role of PD-L2 in modulating the anti-tumor immune response remains controversial (19). PD-L2 inhibits the Crohn-like lymphoid reaction and adaptive immune response during colorectal carcinogenesis (18). In addition, PD-L2 was reportedly upregulated in myeloid-derived suppressor cells with the potential to inhibit anti-tumor immunity and promote tumor growth (45). Previous analyses of The Cancer Genome Atlas (TCGA) dataset revealed that the expression of PD-L2, rather than PD-L1, was positively associated with immune-related gene expression in renal cell carcinoma and lung squamous cell carcinoma (46). Furthermore, PD-L2 was expressed independently of PD-L1 expression, providing limited value for the prediction of anti-PD-1/PD-L1 therapy responses during cancer treatment (19). Although the constitutive expression and binding affinity of PD-L2 are low (24, 47, 48), our findings support that PD-L2 may play an important role in lung carcinogenesis.

Positive correlations between Mycobacterium tuberculosis infection and lung cancer risk were previously reported (49, 50). TB infection can cause chronic inflammation, which may not only lead to innate and adaptive immune responses but may also be associated with immune-related gene expression (51). In this study, pulmonary TB infection was an environmental exposure associated with LUAD risk in never-smoking women. Furthermore, PDCD1LG2 SNPs rs12237624 and rs78096119 had a significant correlation with pulmonary TB infection in relation to lung carcinogenesis. The underlying mechanisms potentially bridging the immune response to TB infection with lung carcinogenesis require further investigation, especially in the TB-endemic areas. Importantly, this finding highlights the importance of gene-environment interaction in relation to lung carcinogenesis for never-smokers.

In our study, the risk alleles of PDCD1LG2 rs2381282, rs12237624, and rs78096119 were negatively associated with PD-L2 expression in non-tumor tissue, but not in tumor tissue. The expression and prognostic value of PD-L2 expression in lung cancer have been previously reported (52–54). The interaction between PD-L2 and PD-1 inhibits strong B7-CD28 signals at low antigen concentrations. At high antigen concentrations, the interaction between PD-L2 and PD-1 reduced cytokine production but did not inhibit T cell proliferation (55). The correlation between these PDCD1LG2 SNPs and PD-L2 expression requires further investigation, which might provide further insight into the PD-1/PD-L2 axis in lung carcinogenesis.

The current study has several limitations. First, this multi-center study was hospital-based. The number of participants was considerably smaller than those in population-based studies. Second, no independent data validation was carried out. Since the proportion of never-smokers in most population-based studies on lung carcinogenesis has been relatively small, large studies in never-smokers are necessary to validate the current findings. Third, our healthy controls were recruited from the health examination departments of six hospitals, which may result in a healthy volunteer effect. Therefore, the current results should be interpreted cautiously.

In conclusion, we identified novel PDCD1LG2 SNPs significantly correlated with LUAD risk in never-smoking women. Of note, some of the identified SNPs interacted with pulmonary TB infection in relation to lung carcinogenesis. These findings may help stratify a high-risk population in never-smokers for early detection of lung cancer.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

The protocol of the study was approved by Research Ethics Committee, National Taiwan University Hospital, Taipei, Taiwan; Institutional Review Board, Taipei Veterans General Hospital, Taipei, Taiwan; Chang Gung Medical Foundation Institutional Review Board, Taipei, Taiwan; Institutional Review Board, Taichung Veterans General Hospital, Taichung, Taiwan; Institutional Review Board, National Cheng Kung University Hospital, Tainan, Taiwan; Institutional Review Board, Kaohsiung Medical University Chung-Ho Memorial Hospital, Kaohsiung, Taiwan; Research ethics committee, National Health Research Institutes, Zhunan, Taiwan; Institutional Review Board, China Medical University Hospital, Taichung, Taiwan. The obtained consents from all participants were written in the study. The patients/participants provided their written informed consent to participate in this study.

CH, I-SC, W-CS, K-YC, Y-HT, C-JC, and P-CY conceived the study and designed the experiments. CH, I-SC, K-YC, L-HC, S-KL, H-HH, and P-CY contributed to study design and statistical analyses. H-CL, W-TF, and S-SJ contributed to the bioinformatics analysis. CH, W-CS, G-CC, K-YC, Y-MC, M-SH, Y-HT, and P-CY contributed reagents and materials. S-KL, L-HC, and K-YC wrote the first draft of the manuscript. C-YC contributed to the collection of tumor specimens for eQTL analysis. W-CS, G-CC, K-YC, Y-MC, M-SH, Y-HT, and P-CY contributed to the recruitment of subjects. All authors contributed to the article and approved the submitted version.

The GELAC study was granted by the National Health Research Institutes (NHRI) and by the Ministry of Sciences and Technology of Taiwan (grant no. MOST 106-2319-B-400-001).

S-KL received honoraria for speech from Roche, AstraZeneca, Pfizer, Merck Sharp & Dohme, Novartis, and Boehringer Ingelheim. K-YC received honoraria for speech from Pfizer, Novartis, Merck Sharp & Dohme, AstraZeneca, Roche, Boehringer Ingelheim, Eli Lilly, Chugai Pharmaceutical, and Bristol-Myers Squibb, as well as travel/accommodation/meeting expenses from Merck Sharp & Dohme, Chugai Pharmaceutical, and Boehringer Ingelheim. Y-MC served on advisory boards of Merck Sharp & Dohme, Ono Pharmaceutical, Astra-Zeneca, Roche, Boehringer Ingelheim, and Bristol-Myers Squibb.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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